Vaccination remains the most effective method of preventing infectious diseases. The World Health Organization (WHO) reports that licensed vaccines are currently available to prevent or contribute to the prevention and control of twenty-five infections (World Health Organization, Global Vaccine Action Plan 2011-2020, Geneva, 2012).
However, current approaches to the delivery of peptide vaccine antigens often rely on technology that is limited in rapid production capability and associated engineering parameters to influence the type, duration, and potency of an immune response. Whilst some vaccine strategies effectively employ protein antigen, custom peptides often lack immunogenic potential and require extensive adjuvanting, and may be most useful as in vitro screening tools.
Recently, RNA has emerged as an attractive antigen vector. mRNAs have been used in mouse models to demonstrate the immunotherapeutic potential of short (<30 aa) neoantigen sequences (Bhadury, et al., Oncogenesis 2, e44, doi:10.1038/oncsis (2013). However, as a vector for immunization, pure mRNA has been investigated with varying degrees of success, particularly in the field of cancer immunotherapy. Administration of naked mRNA can confer anti-tumor immunity when injected directly into lymph nodes (Kreiter, et al., Cancer Res 70, 9031-9040, (2010); Van Lint, et al. Cancer Res 72, 1661-1671, (2012)). Large, replicating RNAs (repRNAs) have also been developed for delivery of vaccine antigens to cells. RepRNA translates and replicates by interacting with the ribosomal machinery of the host cell. Thus, RepRNA provides the template for increasing the number of RNA molecules translating, which in turn increases the rounds of antigen production to elicit prolonged antigen expression relative to an mRNA (McCullough, et al., Molecular Therapy-Nucleic Acids, 3, e173(2014).
However, purified RNAs are notoriously unstable and are extremely vulnerable to degradation, for example, by nucleases, hydroxyl radicals, UV light, and Mg2+-mediated inline attack. Further, the limited translocation across the cell membrane and a substantial liver clearance severely limits the potential applications for RNA-based pharmaceutical compounds such as siRNAs, mRNAs and especially large replicating RNAs. Both the translation and subsequent replication of RepRNA render it particularly sensitive to RNase, which can easily destroy ribosomal entry or gene translation. Thus, delivery of intact, functionally-viable RNA molecules to the intracellular remains a central challenge to the therapeutic application of RNA-based technologies.
Live and attenuated virus-based vaccines, such as non-infectious virions and virus-like particles (VLPs) have been developed with some degree of success. Unfortunately, current live and attenuated virus-based vaccine production methods require long production times. Fertilized egg-based methodology and newer cell bioreactor methods require lead times of months. For production, VLPs also depend on cultured cells. Gene vaccines administered in the form of virus-like particles based on adenovirus, AAV, CMV, rVSV and various alphaviruses, are of limited use due to pre-existing or induced anti-vector immunity, which precludes repeated administration.
The ability to deliver nucleic acids and proteins to mammalian cells has also been demonstrated with biomaterial-based nanoparticles, such as “Polyplexes” of cationic polymers (CPs). However, CP polyplexes and associated nucleic acids are often destabilized by salts and serum components, and can break apart or aggregate in physiological fluids (Al-Dosari, et al. AAPS J. 11, 671-681 (2009); Tros de Ilarduya, et al. Eur. J. Pharm. Sci. 40, 159-170 (2010)) and are considered inefficient as vehicles for the in vivo delivery of encapsulated agent to cells. Further, many cationic polymers exhibit cytotoxicity (Tros de Ilarduya, et al. Eur. J. Pharm. Sci. 40, 159-170 (2010); Gao, et al. Biomaterials 32, 8613-8625 (2011); Felgner, et al. J. Biol. Chem. 269, 2550-2561 (1994); Kafil, et al. Biolmpacts 1, 23-30 (2011); Lv, et al. J Contr. Rel. 114, 100-109 (2006)).
Various nanoparticle formats have demonstrated efficacy through intradermal (Hoerr, et al., Eur J Immunol, 30, 1-7 (2000)), intra-splenic (Zhou, et al., Hum Gene Ther, 10, 2719-2724 (1999)), subcutaneous (Pollard, C. et al., Mol Ther, 21, 251-259 (2013)), intravenous (Hoerr, et al., Eur J Immunol, 30, 1-7 (2000); Mockey, et al., Cancer Gene Ther, 14, 802-814 (2007)) and even intranasal (Phua, et al., Sci Rep, 4, 5128, (2014)) routes of administration. However, few such approaches have graduated to clinical trials. While correlates of immune protection in humans have been reported, clinical efficacy has been disappointing (Weide, et al., J Immunother, 31, 180-188, (2008); Weide, et al., J Immunother, 32, 498-507, (2009); Rittig, et al., Mol Ther, 19, 990-999, (2011); Kreiter, et al., Curr Opin Immunol, 23, 399-406, (2011)). The administration of protamine-complexed mRNA has shown signs of success in intra-dermally immunized murine, ferret, and porcine models of influenza infection (Petsch, et al., Nat Biotechnol, 30, 1210-1216, (2012)).
Large RNA molecules, such as repRNA-based therapeutics, present additional problems for effective in vivo delivery. RNA molecules are susceptible to intracellular degradation when left unmodified, mRNA expression is transient, and translational repression due to inherent immunogenicity of the RNA itself (Kariko, et al., J Biol Chem, 279, 12542-12550, (2004); Pichlmair, et al., Science, 314, 997-1001, (2006); Levin, et al., J Biol Chem, 256, 7638-7641 (1981)), all limit efficacy. Immunogenicity and/or toxicity of the delivery compound used to deploy the vaccine is an additional complication. Cationic lipids, efficacious in some applications, (Geall, et al., Proc Natl Acad Sci USA, 109, 14604-14609, (2012)) are toxic when used at higher doses and if incompletely complexed (Hofland, et al., Proc Natl Acad Sci USA, 93, 7305-7309 (1996); Li, et al., Gene Ther 5, 930-937, (1998); Lv, et al., JControl Release, 114, 100-109, (2006). They depend on a high positive zeta potential for efficient delivery which can become a limiting factor due to neutralization in serum in vivo (Mirska, et al., Colloids Surf B Biointerfaces, 40, 51-59 (2005)). Furthermore, cationic lipids are immunogenic, which can limit transgene expression and raises jeopardizing safety concerns (Henriksen-Lacey, et al. Mol Pharm, 8, 153-161, (2011)). IFN production in response to mRNA can indeed limit efficacy of mRNA-based vaccines (Pollard, et al., Mol Ther, 21, 251-259 (2013)). Lipid nanoparticles created using cationic lipids also generally require additional stabilizing excipients in their formulation, raising cost and complexity of the final product.
There exists a need for improved systems for effectively delivering an encapsulated agent, such as intact genetic material, into the cells of a subject.
Therefore, it is an object of the invention to provide compositions and methods for intracellular delivery of nucleic acids, proteins and small molecules, by delivery vehicles that exert minimal or no immunogenicity and cytotoxicity.
It is a further object of the invention to provide methods and compositions for the simultaneous delivery of two or more repRNA molecules to the interior of cells.
It is a further object of the invention to provide compositions, methods, and devices for sustained expression of exogenous genes by target host cells.
It is a further object of the invention to provide methods and compositions for the delivery of therapeutic molecules into the cells of subject.